Phospholipase C-.alpha. (hereinafter referred to as PLC-.alpha.) has conventionally been considered to belong to the superfamily of various isozymes of phospholipase C (hereinafter referred to as PLC).
PLC is an enzyme hydrolyzing (hereinafter referred to as a PLC activity) glycerophospholipid and sphingophospholipid; which is known to be present in the spleen, the tunica mucosa interstini tenuis, and the placenta, etc. of mammals and to play an important role in a living body. For example, phosphatidylinositol-specific phospholipase C (PI-PLC), which is widely present in a living body, hydrolyzes phosphatidylinositol 4,5-diphosphate to generate 1,2-diacylglycerol and inositol 1,4,5-triphosphate (Rhee, S.G. et al., Science 244, pp. 546-550 (1989)). There are a number of reports on various isozymes of PLC, for example, as follows: C. F. Bennett et al., Nature 334, pp. 268-270 (1988); Y. Emori et al., J. Biol. Chem. 264, pp. 21885-21890 (1989); M. Katan et al., Cell 54, pp. 171-177 (1988); S. Ohta et al., FEBS Lett. 242, pp. 31-35 (1988); M. L. Stahl et al., Nature 332, pp. 269-272 (1988); P. G. Suh et al., Cell 54, pp. 161-169 (1988); and R. W. Kritz et al., CIBA Found. Symp. 150, pp. 112-127 (1990).
In terms of function, the above-mentioned PLC-.alpha. is not well known; however, the expression thereof is known to increase in a living body being subject to stress and having cancerous tissue.
Regarding the PLC-.alpha. of mammals; those of mice (W. M. Hempel et al., J. Immunol. 146, pp. 3713-3720 (1991)), rats (C. F. Bennett et al., Nature 334, pp. 268-270 (1988)), and bovines (Hirano et al., Extended Abstracts, 4L-23, The 15th Annual Meeting of The Japanese Society of Molecular Biology, 1992), have been reported. The PLC-.alpha. of these mammals does not have significant homology with any of known sequences of the other known PLC family members.
The PLC-.alpha. of the above-mentioned mammals has a conserved amino acid sequence, Trp-Cys-Gly-His-Cys-Lys,[SEQ ID NO:5] at two places. This conserved amino acid sequence is identical with or very similar to the amino acid sequence of an active site for oxidation-reduction activity of protein disulfide isomerase (PDI) and thioredoxin. PDI and thioredoxin are both multifunctional proteins which function as protein disulfide reductase and isomerase, and catalyze the conversion of a thiol group to a disulfide group. PDI of mammals has two conserved sequences in its amino acid sequence, which are homologous to the above-mentioned PLC-.alpha.. Thioredoxin of prokaryotes and eukaryotes has one sequence, Trp-Cys-Gly-Pro-Cys-Lys, [SEQ ID NO:6]in its amino acid sequence which is similar to the conserved sequence as shown above. Cys residues in these sequences are respectively assumed to be active sites of catalytic activity (A. Holmgren, J. Biol. Chem. 264, pp. 13963-13966; R. B. Freedman, Cell 57, pp. 1069-1702).
The inventors of the present invention have found that bovine PLC-.alpha. expressed in E. coli is a secretory protein which does not functionally have PLC activity and has the activity of reducing a disulfide bond of protein such as insulin (Hirano et al., Cloning of bovine PLC-.alpha. and biological significance thereof, Extended Abstracts, 4L-23, The 15th Annual Meeting of The Japanese Society of Molecular Biology, 1992). Thus, PLC-.alpha. is not supposed to belong to the PLC superfamily but to the oxidation-reduction control protein family; and its nomenclature has accordingly been changed to thymuredoxin (Hirano et al. supra). This is supported by the above-mentioned consensus of the sequence.
The oxidation-reduction control protein family including PLC-.alpha., thioredoxin, PDI, etc. is considered to be required for maintaining the normal function of a living body. Hereinafter, findings obtained from various reports on various members of the oxidation-reduction control protein family will be shown.
PLC-.alpha. has its expression level increased in animal cells transformed with oncogenes, v-src gene; therefore, it is considered to be related to carcinogenesis (H. Hirai et al., Genes Dev 4, pp. 2342-2352 (1990), Mol. Cell. Biol. 10, pp. 1307-1318 (1990), Proc. Natl. Acad. Sci. U.S.A. 87, pp. 8592-8596 (1990)).
Human thioredoxin, also called adult T-cell leukemia (ATL) derived factor, is known to induce the expression of interleukin-2 receptor and stimulate the multiplication of cells (Y. Tagaya et al., EMBO J. 8, pp. 757-764 (1989)).
The protein level of thioredoxin is generally high in actively expanding tissue and the mRNA level of thioredoxin increases (S. W. Jones et al., J. Biol. Chem. 263, pp. 9607-9611 (1988)). As one of functional mechanisms maintaining the normal cell morphology and function of the oxidation-reduction protein control family, controls of the relationships between DNA and protein and between proteins through a Cys residue are considered. This is described in the following document incorporated in the present specification for reference.
The binding of fos and Jun heterodimers to DNA, which relates to v-jun genes and v-fos genes having activity as oncogenes, is regulated by the oxidation-reduction control of a conserved Cys residue in a DNA binding region of these two kinds of proteins: thioredoxin can catalyzes this regulation (C. Abate et al., Science 249, pp. 1157-1161 (1991)). Receptor type tyirosinekinase (1tk) is regulated through the change in a cell oxidation-reduction ability, and kinase activity thereof is increased by the formation of a multimer composed of disulfide bonds (A. R. Bauskin et al., Cell 66, pp. 685-696 (1991)).
Although a control factor of a Cys mediated oxidation-reduction ability in vivo has not been identified, PLC-.alpha., thioredoxin, and PDI have a thiol-dependent catalytic activity because of a sequence Trp-Cys-Gly-His-Cys-Lys[SEQ ID NO:] or Trp Cys-gly-Pro-Cys-Lys [SEQ ID NO:6] therefore, there is a possibility that these are control factors.